1. Field of the Invention
The invention relates to the detection and measurement of predetermined substances capable of being bound specifically, using a novel method and combination therefor which provide for rapid qualitative and quantitative determinations in an advantageous manner.
2. Description of the Prior Art and Other Information
Various methods have been developed in the last two or three decades for the determination of a variety of substances capable of being bound specifically, generally immunochemical substances, including antigens, antibodies, haptens, and certain low molecular weight substances. Examples of these methods are:
1. Radioassay techniques PA1 2. Fluoroimmunoassays (FIA) PA1 3. Enzyme immunoassay (EIA); including competitive, enzymometric, double antibody solid phase, sandwich, etc. PA1 4. Lysis-initiating immunoassays (LIA) PA1 5. Latex-particle agglutination (LPA) PA1 6. Charcoal-particle agglutination (CPA) PA1 7. Hemagglutination and Hemagglutination Inhibition Assays (HA), (HIA) PA1 8. Complement Fixation (CF) PA1 9. Counter-immunoelectrophoresis (CIEP) PA1 10 Radial Immunodiffusion and Double Diffusion (RID) PA1 11. Viroimmunoassay (VIA); and PA1 12. Spin immunoassay (SIA) among others. PA1 13. Turbidity (physical assay) PA1 (a) end cap means adapted to slidably receive the covered probe tip end of said light conducting probe, and PA1 (b) suitable reflective surface means effectively disposed near below or touching the bottom of said well for reflecting an effective amount of the illuminating light to the second light conducting means.
a. Competitive protein binding assays PA2 b. Radioimmunoassay (RIA); including competitive, immunoradiometric, sandwich, etc. PA2 (1) having a substantially flat collar with a suitable abutment means adapted to join the top of the microplate well in sealing engagement, but not to allow the end cap means to contact said liquid sample, and PA2 (2) allowing light to be transmitted from the first light conducting means to the well, and PA2 (3) allowing light from the well to be received by the second light conducting means, and
One type of immunochemical test system involves the use of labels: e.g. radioisotopes, enzymes, fluorescent labels, etc. Within these, there are many types of labels useful in assays for the detection and measurement in serum or other media or biologically important or interesting compounds or substances. The administration of most of these tests are hampered by one or more of the following limitations: (1) lack of sensitivity, (2) complexity of the test procedure, (3) instability of reagents, (4) hazardous reagents, (5) impure reagents, and (6) expensive equipment required to perform quantitative and qualitative analysis of the amount of label involved in an immunochemical reaction. For a review of the development and evaluation of immunological methods and their uses as diagnostic tools, reference is made to "Immunology as a Laboratory Tool", by FRANZ PEETOOME, American Journal of Technology, 37(2), pp. 445-469 (1971), incorporated herein.
There are other immunochemical test systems which do not use labels for a means of detection; some of these are the so-called "agglutination" tests, wherein the analysis depends on the measurement of certain electromagnetic radiation properties of the liquid samples containing immunochemical constituents, with the measured properties depending on whether or not an immunochemical reaction has taken place. A pioneer reference in this area of technology is Schuurs, U.S. Pat. No. 3,551,555 (1970). See also Price et al, U.S. Pat. No. 4,066,744 (1978); both patents are incorporated herein by reference. Examples of these "aggultination" tests include the so-called "NOSTICON" latex tests by Organon Incorporated (West Orange, N.J.), including PREGNOSTICON.RTM., RHEUMANOSTICON.RTM., AND GONOSTICON.RTM. latex agglutination or latex agglutination inhibition slide and DRI-DOT.RTM. Tests (distinguishable from the "NOSTICON" erythrocyte agglutination inhibition tests).
Both labeled and unlabeled immunochemical testing may employ various devices to separate immunochemical constituents which have reacted from non-reacted immunochemical constituents and from substances irrelevant to the test. For example, some EIA patents require separation through the use of one component in the antigen-antibody reaction being in an "insolubilized" phase for separation--see Schuurs and co-workers in U.S. Pat. Nos. 3,654,090; 3,791,932; 3,850,752; 3,839,153; 3,879,262; 4,016,043 and U.S. Pat. No. Re. 29,169.
Another method does not require separation of free and bound label because the assay depends on the inhibition or activation of the enzymelabel by antibody binding (e.g., the EMIT type system of Syva Corporation of Palo Alto, CA, for EIA and FRAT, or "free radical assay technique", for SIA)--see U.S. Pat. Nos. 3,880,715; 3,852,157; 3,875,011; 3,935,074; and 3,905,871, and an article by Kenneth S. Rubenstein et al in "Homogenous Enzyme Immunoassay, a New Immunochemical Technique", Biochemical and Biophysical Research Communications 47, No. 4, 846-851 (1972) (all incorporated herein by reference). There are examples wherein an insolubilized phase is not employed and the assay depends on inhibition or activation of the enzyme label by antibody binding. See also G. Brian Wisdom, "Enzyme Immunoassay", Clinical Chemistry 22/8, 1243 (1976).
Radioimmunoassay (RIA) is now considered a classical and well-known technique for detecting antigens at very low concentrations. It is based upon the competition between radio-labeled and unlabeled antigen for a fixed, limited amount of antibody, as described by R. Yalow and S. Berson in J. Clin. Invest., 39 1157 (1960). The amount of unlabeled antigen influences the distribution of the labeled antigen in antibody-bound (B) and antibody-free (F) labeled antigen, i.e., the more that unlabeled antigen is present, the less the labeled antigen is able to combine with the antibody. In order to obtain conclusive results from the distribution, a good separation between B and F must be made. Methods used for this purpose are, for instance, chromatoelectrophoresis, described by S. Berson and R. Yalow in The Hormones, edited by G. Pincus et al, Academic Press, New York (1964), vol. IV, 557, or insolubilization of the antibodies. This insolubilization can be achieved by chemical means (cross-linking or covalent binding to an insoluble carrier) or by physical methods (absorption to an insoluble carrier).
Of the limitations cited above, a most serious limitation until recently has been lack of adequate sensitivity to detect some antigens. In general, three levels of sensitivity are recognizable. Low sensitivity techniques, where materials detected and measured exist in microgram/milliliter quantities, include RID, CF, CEP, CPA, and LPA. Intermediate sensitivity techniques, where microgram/milliliter to nanogram/milliliter quantities of materials may be measured, include HIA, HA, CF, FIA, SIA, VIA, and EIA. Until recently only RIA was able to measure with ultrasensitivity the picogram/milliliter to femtogram/milliliter region.
Many of the techniques listed above require that some form of physically or chemically identifiable label be attached to reagents in the assay system in order that the result of a test can be detected. RIA, FIA, EIA, LIA, VIA, and SIA all fall into this category. Radioactivity, fluorescent moieties, enzymes, complement, viruses, and electron-spin labels are used respectively to generate some form of end-point signal. The sensitivity with which these labels can be detected directly and fundamentally affects the useful ranges of the test systems using them.
The ability to measure the amount or concentration of a label depends upon: (a) the nature of the signal that it generates; (b) the ability of the detector to differentiate the proper signal from the background or interfering signals; (c) the intensity of signal available per unit amount of marker molecule, i.e., the specific activity of the label. With radioactive labels, heretofore the most popular label in use, the signal is decay radiation. Because of the energetic properties of the emissions generated, some of which are penetrating, radioactive decay can be detected easily. Generally, modern counting equipment can be effectively applied to measure the radioactive material. Furthermore, there is a range of specific activities offered by isotopes currently used for tagging.
As noted, up to the present time, the radioimmunoassay (RIA) method in its various forms has been the most sensitive system available. The RIA method, unfortunately, has several serious disadvantages, including the requirement of special equipment, trained staff, the need for extra safety measures to protect against harmful radiation, special licensing, controlled radioactive wastes disposal and the continuous disappearance of labeled compound by radioactive decay. In some instances radiation detection is inefficient. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 in an article by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodies and Immunological Assay Systems", Lancet, II, 492 (1968), and Nature 219, 169 (1968). No procedural details were provided, the article offered only the general idea, leaving it to future workers to determine the basic steps and to perform the extensive experimentation needed to establish a practical operative enzymatic immunoassay method.
More recently, methods for detecting and measuring immunochemical substances have been developed in which, in lieu of a radioactive isotope, immunochemical substances have been labeled with other materials which can be detected by various techniques, e.g., optical and electronic instrument methods. One useful group of materials is enzymes which, because of the great number of analytical permutations, has created a whole family of techniques known collectively as enzyme immunoassay (EIA) techniques.
Among the more recent patents issued that are representative of the state of the art in the detection and measurement of immunochemical substances are, as recited, U.S. Pat. Nos. 3,654,090, issued Apr. 4, 1972; 3,666,421, issued May 30, 1972; 3,791,932, issued Feb. 12, 1974; 3,839,153, issued Oct. 1, 1974; 3,850,752, issued Nov. 26, 1974; 3,879,262, issued Apr. 22, 1975; 4,016,043, issued Apr. 5, 1977; and U.S. Pat. No. Re. 29,169, reissued from U.S. Pat. No. 3,791,932, Apr. 5, 1977, all incorporated by reference herein. See Examples II and III for use of the novel apparatus of our invention in enzyme immunoassays.
A specific example of a recent latex agglutination inhibition method is a qualitative in vitro test for determining the presence of human chorionic gonadotropin (HCG). HCG is a hormone that is characteristic of pregnancy and may be found in the urine of a pregnant human. An antiserum specific to HCG can be prepared from rabbits immunized with HCG to produce the antibody.
According to the PREGNOSTICON.RTM. Slide Test, if the antiserum is mixed with latex that has been sensitized (coated) with HCG, agglutination of the latex occurs. If, on the other hand, the antiserum is mixed with a sample of urine containing HCG, i.e., from a pregnant person, the antiserum is neutralized, and upon subsequent mixing of the antiserum-urine mixture with the HCG-sensitized latex, the agglutination of the latex is inhibited. The latex appears as a milky homogenous suspension, its agglutination having been inhibited. This is a positive test for pregnancy.
Although usually a positive or negative result can be determined by a lack of agglutination or agglutination of the latex, respectively, a maximum inhibition of agglutination may not occur in the early stages of pregnancy when the concentration of HCG in the urine has not increased above a certain threshold level which can be detected by this method. The sensitivity of the described pregnancy test is normally such that the concentration levels of HCG are usually sufficiently elevated by the twelfth day after menstruation fails to occur that the HCG can be detected with the test. If the result of the test is inconclusive, the test must be conducted again in another week or two, to allow sufficient time for an increase in the HCG concentration in the urine to detectable levels. Of course, this is very undesirable from a diagnostic viewpoint, since it is often important to be able to determine the existence of pregnancy at the earliest stages.
The above-described test is qualitative in nature, giving either a positive or a negative test for some threshold concentration of HCG. If HCG is detected in a urine sample, a more quantitative determination of the concentration of HCG in the sample can be made by conducting a series of tests on a series of systematic serial dilutions (commonly referred to in the art as a "dilution series") of the urine samples. Of course, the necessity of conducting a series of tests to determine the concentration of HCG in a single urine sample is time-consuming and costly.
In an available embodiment, the foregoing technique for qualitatively detecting the HCG antigen characteristic of pregnancy is known as the PREGNOSTICON.RTM.-Slide Test (Organon Inc., West Orange, N.J.).
The same general agglutination process principles underlying the PREGNOSTICON.RTM.-Slide Test (and erythrocyte test) can be applied to the determination of other immunochemical substances which can be specifically bound, such as antigens i.e., those associated with gonorrhea (GC), rheumatoid factor (RF), etc., and antibodies such as those specific to GC and RF and so forth. It is now desirable in the art to provide a test for determining immunochemical substances which would both detect and quantitatively measure the presence of such specifically-bindable immunochemical substances rapidly and at low concentrations, to thereby insure early diagnosis.
There are two types of measuring concepts commonly used in the industry: (a) the first (hereinafter "Concept I"), which employs the concept of light-scattering, including scattering by suspended particles, which block a certain amount of light, so that light which is not absorbed or reflected in a non-detector direction by the particles in what is actually measured (this includes nephelometric methods); and (b) a second, which involves the measurement of the absorption of light by molecules in solutions or "Concept II". Our invention may employ either Concept I or II, but generally employs Concept II.
The use of light-scattering methods of Concept I in analyzing the electromagnetic properties of various substances is well known, and such photometric methods can be used in the analysis of immunochemical substances. There are many different embodiments in which light-scattering Concept I has been used, but, in most cases, the instrumentation required is very sophisticated and expensive. One reason for this is that such instruments typically include a number of lens systems and complicated mechanisms for positioning the cuvette containing the substance being determined, for example, the BRICE-PHOENIX Model OM-2000 Light Scattering Photometer (Virtis Co., Gardiner, N.Y.); SCIENCE SPECTRUM Differential Light-Scattering.RTM. Photometer (Santa Barbara, Cal.). For example, U.S. Pat. No. 3,036,492 issued May, 29, 1962, describes a complex adjustable specimen chamber for determining the light transmittance properties of a sample at varying angles. Another example is U.S. Pat. No. 3,918,817 issued Nov. 11, 1975, which describes a turbidimeter, a particular type of photometer, including a special thermal insulating housing and using a glass test tube of rectangular cross-section. Buffone, "Improved Nephelometric Instrumentation", Laboratory Management, April, 1977, describes at page 19 a nephelometer, a similar type of photometer, using an incandescent lamp with filters to produce a band of radiation between 450 and 650 mn.
Much of the literature relating to Concept I has been concerned with textile quality control techniques. For example, textile color analyzers involving instrument head using a plurality of fiber-optic bundles positioned to receive diffuse light reflected from the textile samples have been devised, as described in U.S. Pat. No. 3,986,778 issued Oct. 19, 1976, and U.S. Pat. No. 3,999,860 issued Dec. 28, 1976.
It is interesting to note that Concept I light-scattering photometric agglutination diagnostic methods for determining particular substances have in the past typically required measurement at a particular wavelength, that is, essentially monochromatic light (we believe that these teachings may well be valid to some extent for non-dispersed enzyme immunoassays not requiring the use of particles, but not for agglutination tests).
For instance, in Blume and Greenberg, "Application of Differential Light Scattering to the Latex Agglutination Assay for Rheumatoid Factor" Clinical Chemistry, Vol. 21, No. 9, 1975, page 1235 et seq., it is disclosed that in the technique of differential light scattering it is essential that the light source be highly monochromatic, such as, e.g., that produced by a helium/neon-laser (632.8 nm). The requirement of essentially monochromatic light in photometric determinations is again described by Lichenbelt, Pallmarnanobaran, and Wiersema, "Rapid Coagulation of Polystyrene Latex in a Stopped Flow Spectrophotometer", Journal of Colloid & Interface Science, Vol. 49, No. 2, 1974, page 281 et seq., and Dininno and McCandless, "Agarose Medium Turbidimetric Assay for Cross-Reacting Antigens", Journal of Immunological Methods, Vol. 17, 1977, pages 73-79. See also Flurometry Reviews, March 1969 (monthly bulletin of Turner Inc., Palo Alto, CA.
Surprisingly, it has now been found in a major invention by O'Conner in Ser. No. 909,862, filed May 26, 1978, and in a continuation-in-part application thereof (attorney docket OR14200A), filed Aug. 9, 1978, incorporated by reference herein, that for the detection and measurement of overall "average" agglutination of certain insolubilized latex particles in suspension, as opposed to the measurement of the distribution of clump sizes i.e., non-agglutinated or agglutinated latex particles, the requirement for amonochromatic, incident light source is illusory. It has also been found in Ser. No. 909,862 that in agglutination tests the widest spectral band of incident light available, whose upper wavelength limit being equal to or less than the mean diameter of the insolubilized particles in the suspension of interest (the value of which may be expresses herein in nanometers or microns), is preferred for optimum detection and measurement sensitivity. The use of wide-band spectral filters, commonly known as low-pass optical filters, in association with an appropriate light source, is indeed unique and novel for the aforementioned applications.
On the other hand, for commercial non-dispersed EIA tests Concept II is made as no particles are employed, and one should use light having a narrow band width centered around the absorbance peak of the substance to be deleted, for example equal to or narrower than 370-430 nm for solutions of the substrate o-phenylenediamine when used with the enzyme alkaline phosphatase.
Testing of agglutination and EIA samples in cuvettes and test tubes has been performed in the prior art in Concept II equipment by complex machinery having optical lens assembles wherein light is passed through the cuvettes from above to detecting means below requiring expensive automated equipment to move the light source from well to well as well as an optical lens assembly. Some systems had great variations on readout because of the optical variations inherent in the measuring small volumes in small test tubes or cuvettes by conventional means. See LABSOURCE RP-800.TM. Photometer by PBI Electro-optics Inc., West Westbury, Mass.; FINNPIPETTE.TM. Analyzer System by Labsystems Oy, Helsinki 81, Finland (a forward-scattering system); Olli 3000 Clinical Chemistry Analyzers.TM. by Olli Medical Electronics Co., Kivenlahti, Finland; and the Fixed Dual Wavelength Microcomputer Controlled Visible Spectrophotometer by Cooke Laboratory Products, Division of Dynatech Labs Inc., Alexandria, VA. Some concept II attempts have been made using "side-to-side" measurement using low-cost test tubes in colorimeters, such as the SPECTRONIC 20 by Bausch & Lomb (Rochester, N.Y.), which requires a long amount of time for hand insertion and removal of the individual cuvettes, and a large amount of solution to be measured, therefore making measurements of small volumes (i.e., those in microplates) impossible.
To our knowledge the prior art systems did not employ fiberoptic bundles, a reason we now see by hindsight for the necessity of prior art complex systems to either (1) move each cuvette receiving light (and transmitting it to photometer means for detection) relative to a stationary light source means, or (2) move the light souce/light receiving means relative to the stationary cuvette. In either event, economical standard microplates for use in enzyme immunoassay (for example, MICROTITRE.RTM. plates manufactured by Cooke Laboratory Products Div., Dynatech Labs Inc., supra) could not be employed. Visual evaluation of microtitre plates led to subjective and nonreproducible reports of testing. A need therefore existed for an inexpensive manual reader for microplates which would avoid the use of optical lens assemblies and transport systems for light transmission receiving means, would provide a stable readout, and would provide rapid inexpensive evaluation of samples.
In the prior art methods involving the colorimetric determination of liquids using fiber optic probe colorimeter, various [acid resistant and stainless steel or glass] probe tip means has been employed substantially as shown in FIG. 2.
See the Brinkmann Instruments Inc. (Westbury, N.Y.) adapters-Digital probe Colorimeter PC 600D Catalogue, Nos. 20-20-932-1, 20-20-930-5, 20-22-010-4, 20-20-890-2, etc., all requiring contact with the sample medium, so that the tip had to be washed when each sample was tested to avoid contamination, requiring undue testing time. If the probe colorimeter was to be used in the relevant EIA/agglutination art, a need arose to employ a probe tip means of a fiber optic probe colorimeter that could be used to test numerous samples in the wells a microtitre plate quickly and accurately, without washing.
A number of references in distant arts are directed to an optical probe arrangement, employing fiberoptic "light pipes" used in a measuring or testing capacity, c.f. U.S. Pat. Nos. 3,068,742 (Hicks, Jr.); 3,164,663 (Gale); 3,235,672 (Beguin); 3,383,979 (Gibson); 3,493,304 (Rovner); 3,566,083 (McMillin); 3,885,878 et al (Ishak); 3,906,241 (Thompson); and 4,033,698 (Demsky); 4,039,845 (Oberhiinsli). U.S. Pat. No. 3,885,878 and U.S. Pat. No. 4,033,698 both are directed to a measuring apparatus in which suitable fitting or adapter for a positively locating associated fiber optic means is employed. In U.S. Pat. Nos. 3,068,742 and 3,906,241, fiberoptic probes are employed in colorimetric devices. Weinstein in U.S. Pat. No. 3,932,763 discloses a detector for tubular transport articles employing a light beam directed across an opening in a housing, along a path offset from a diameter of that opening. Mudd in U.S. Pat. No. 3,773,426 discloses a device for detecting bacterial growth in a plurality of dilutions wherein a test tray containing a plurality of test wells, each well having a different dilution, is inserted in a frame so that light passes through each well and onto a photo-transistor. If bacterial growth is present in the test well, the light is attenuated, and the drop detected electronically, and punched onto a card. A row of sensors comprising a phototransistor for each of eight dilutions simultaneously reads the samples, light being conducted to each sensor by means of fibre optic bundles. Komarniski in U.S. Pat. No. 3,627,431 discloses a colorimeter wherein a number of samples can be read simultaneously; the instrument incorporates a filter so that the optical density of all samples can be compared on a gray scale. See also U.S. Pat. Nos. 3,518,009 (Shamos); 3,566,083 (McMillin); 3,656,833 (Wallace); 3,773,424 (Selgin); 3,781,092 (Susman); 3,786,266 (Reid); 3,488,156 (Good); and 4,029,391 (French).
It is also interesting to note that recent art complex photometers which have been attempted to be employed for polystyrene microplates in enzyme-linked immunosorbent assays (ELISA) have insisted on employing flat-bottom plates. See E. J. Ruitenberg et al, "Direct Measurement of Microplates and Its Application to Enzyme-Linked Immunosorbent Assay", 3(5) J. CLINICAL MICROBIOLOGY 541-542 (1976).
The preceding discussion illustrates that a need exists for (a) a new improved method and (b) a low cost, simple to use apparatus therefor for making rapid, accurate, and economical measurements of specific binding substances, and in particular, immunochemical substances in emzyme immunoassay agglutination and other colorimetric medical-diagnostic laboratory tests, employing inexpensive disposable microplates having scratches and small defects normally found in the science laboratory.